PCR (Polymerase Chain Reaction) is a molecular biological method for the in-vitro amplification of nucleic acid molecules. The PCR technique is rapidly replacing many other time-consuming and less sensitive techniques for the identification of biological species and pathogens in forensic, environmental, clinical and industrial samples. PCR using microfabricated structures promises improved temperature uniformity and cycling time together with decreased sample and reagent volume consumption.
An efficient thermal cycler particularly depends on fast heating and cooling processes and high temperature uniformity. Presently, microfabricated PCR is preferably carried out on a number of samples during a single thermal protocol run. It is a great advantage if each reaction chamber can be controlled to have an independent thermal cycle. This makes it possible to run a number of samples with independent thermal cycles simultaneously (parallel processing). The first work on multi-chamber thermal cyclers fabricated multiple reaction chambers by silicon etching. Although separate heating elements for every reaction chamber can be realized, it was impossible in these designs to eliminate thermal cross-talk between adjacent reaction chambers during parallel processing because of limited thermal isolation between reaction chambers. As a result, multiple chambers having independent temperature protocols could not be used. Additionally, temperature uniformity achieved inside the reaction chamber was xc2x15 K in this thermal isolation and heating scheme.
Integration of the reaction chamber with micro capillary electrophoresis (CE) is also an interesting subject, in which small volumes of samples/reagents will be required both for PCR and CE. Again, a high degree of thermal isolation is very important particularly where various driving/detection mechanisms prefer a constant room temperature substrate.
A number of microfabricated PCR devices have been demonstrated in the literature. Most of them were made of silicon and glass, while a few others were using silicon bonded to silicon. On-chip integrated heaters and temperature sensors become important in the accurate control of the temperature inside these small reaction chambers. Good thermal isolations have been proved promising for quick thermal response. Micro reaction chamber integrated with micro CE was only demonstrated where no PCR thermal cycling was performed (only slowly heated to 50xc2x0 C. in 10-20 seconds and held for 17 minutes). Parallel processing microfabricated thermal cyclers with multi-chamber and independent thermal controls have not yet been reported.
A routine search of the prior art was performed with the following references of interest being found: Northrup et al. (U.S. Pat. No. 5,589,136 December 1996), Northrup et al. (U.S. Pat. No. 5,639,423, U.S. Pat. No. 5,646,039, and U.S. Pat. No. 5,674,742), and Baier Volker et al, in U.S. Pat. No. 5,716,842 February 1998), did early work on multi-chamber thermal cyclers fabricated by silicon etching. Baier et al. (U.S. Pat. No. 5,939,312 August 1999) describe a miniaturized multi-chamber thermal cycler. This latter reference includes the following featuresxe2x80x941. multiple chambers placed together within a silicon block from which they are thermally isolated. This approach works against fast cycling because of slow cooling by the chambers. 2. The chambers are packed together very closely, with minimal thermal isolation from one another, so all chambers must always to be thermally cycled with the same thermal protocol. The individual chambers were not subject to independent thermal control of multi-chambers. 3. Baier""s units have thin-film heaters that cover the whole bottom of the chamber (as in conventional heating designs). 4. Baier""s apparatus is limited to the chambers, no micro-fluidic components (valves, fluidic manipulation, flow control, etc.) being included.
Micro-fabricated PCR reaction chambers (or thermal cyclers) have been reported in the technical literature by a number of experimenters, including: (1). Adam T. Woolley, et al, (UC Berkeley), xe2x80x9cFunctional Integration of PCR Amplification and Capillary Electrophoresis in a Microfabricated DNA Analysis Devicexe2x80x9d, Analytical Chemistry, Vol. 68, pp. 4081-4086, (2). M. Allen Northrup, et al, (Lawrence Livermore National Lab, UC Berkeley, Roche Molecular Systems), xe2x80x9cDNA Amplification with a microfabricated reaction chamberxe2x80x9d, 7th Intl. Conf. Solid-State Sensors and Actuators, pp. 924-926, (3). Sundaresh N. Brahmasandra, et al, (U. Michigan), xe2x80x9cOn-Chip DNA Band Detection in Microfabricated Separation Systemsxe2x80x9d, SPIE Conf. Microfuidic Devices and Systems, Santa Clara, Calif., September 1998, SPIE Vol. 3515, pp. 242-251, (4). S. Poser, et al, xe2x80x9cChip Elements for Fast Thermocyclingxe2x80x9d, Eurosensors X, Leuven, Belgium, September 96, pp.11971199. The latter showed promising results for use of well thermal isolation as a means for achieving quick thermal response.
Also of interest, we may mention: (5). Ajit M. Chaudhari, et al, (Stanford Univ. and PE Applied Biosystems), xe2x80x9cTransient Liquid Crystal Thermometry of Microfabricated PCR Vessel Arraysxe2x80x9d, J. Microelectromech. Systems, Vol. 7, No. 4, 1998, pp. 345-355, (6). Mark A Burns, et al, (U Michigan), xe2x80x9cAn Integrated Nanoliter DNA Analysis Devicexe2x80x9d, Science 16, October 1998, Vol. 282, pp. 484-486, and (7). P. F. Man, et al, (U. Michigan), xe2x80x9cMicrofabricated Capillary-Driven Stop Valve and Sample Injectorxe2x80x9d, IEEE MEMS""98 (provisional), pp. 45-50.
It has been an object of the present invention to provide a microfabricated thermal cycler which permits simultaneous treatment of multiple individual samples in independent thermal protocols, so as to implement large numbers of DNA experiments simultaneously in a short time.
A further object of the invention has been to provide a high degree of thermal isolation for the reaction chamber, where there is no cross talk not only between reaction chambers, but also between the reaction chamber and the substrate where detection circuits and/or micro fabricated Capillary Electrophoresis units could be integrated.
Another object has been to achieve temperature uniformity inside each reaction chamber of less than xc2x10.5 K together with fast heating and cooling rates in a range of 10 to 60 K/s range.
These objects have been achieved by use of a thermal isolation scheme realized by silicon etch-through slots in a supporting silicon substrate frame. Each reaction chamber is thermally isolated from the silicon substrate (which is also a heat sink) through one or more silicon beams with fluid-bearing channels that connect the reaction chamber to both a sample reservoir and a common manifold. Each reaction chamber has a silicon membrane as its floor and a glass sheet as its roof. This reduces the parasitic thermal capacitance and meets the requirement of low chamber volume. The advantage of using glass is that it is transparent so that sample filling and flowing can be seen clearly. Glass can also be replaced by any kind of rigid plastic which is bio- and temperature-compatible.